BrdU Staining Protocol
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Supplies:
Ethanol 100 USP (highest quality)
FACS Staining Buffer (1XPBS w/ 3 calf serum, 0.05 azide--filtered) ―Dilute staining antibodies in Buffer
DNAse (Sigma D-5025, Bovine Pancreas)
RNase (Boehringer, 25 mg bovine pancrease)
Anti-BrdU-FITC (Becton Dickinson or Phoenix Flow)
0.15 M NaCl, 1.5 M NaCl
10 Paraformaldehype (kept as stock in -80°C)
Tween
1M MgCl2
FACS Tubes
Protocol:
Cells in 96 well FACS plate
- Block with 24G-2
-
Surface stain cells as usual
-Omit fourth channel labeled antibodies on all stains; EtOH destroys APC - Prepare tubes from which to transfer EtOH drop wise (1.2 ml EtOH on ICE)
- Resuspend cells from 96 well place with 100 µl 0.15M NaCl (cold)
- Transfer to FACS tubes ON ICE. Add 400 µl 0.15M NaCl to each tube
-
Vortex at 1/3 speed and add EtOH with pasteur pipette at 1 drop per second.
This is a critical step... do not add EtOH too quickly - Incubate on ice for 30 minutes
- Spin 10 minutes @ 2000 RPM, 4° C
- Dump and shake liquid into waste
- Using repeat pipetter, squirt 1 ml FACS staining buffer into each tube
- Spin 10 minutes and dump as before (step 8)
-
Add 1 ml 1 paraformaldehyde + 0.05 Tween 10
-For 20 ml:
2.0 ml 10 paraformaldehyde
10 µl Tween-20 - Incubate at room temperature for 30 minutes
- Incubate on ice for 30 minutes
-
Spin and dump as before (step 8)
Add 1 ml DNAse (0.15M NaCl + 4.2mM MgCl + 100 Kunitz units/ml DNAse)
-For 50 ml:
46.5 mL dH20
200 ul MgCl2 (1M stock)
1500 uL NaCl (5M stock)
100 Kunitz units Dnase (volume depends on activity of batch)
Incubate for 30 minutes @ 25° - Spin 10 min. and dump as before (step 8)
- Transfer cells from FACS tubes to 96-well plate. Wash once with staining media.
- Block with 10 rat serum. Incubate 15 minute on ice. Spin and dump as before (step 8: it is critical to spin at high speed once the cells have been fixed with EtoH/ PFA since they become less dense).
- Add anti-BrdU-FITC or biotin (1:20 dilution for Phoenix flow).
- Pipette up and down to resuspend pellet. Incubate for 30 minutes on ice (or overnight at 4°C).
- Wash and dump as before. Transfer cells into FACS tubes.